Our long term objectives are to elucidate the mechanism of intracellular parasitism in leishmaniasis. By studying the cell biology of host-parasite interactions in vitro, we have already answered in part the questions of how leishmanias attach themselves to the macrophage, and how they subsequently gain intracellular entry, survive and multiply. Intracellular promastigote to amastigote transformation in leishmaniasis is a crucial event which determines not only the outcome of infection but also the survival of leishmanias. Particularly amenable to investigations of this transformation, not achievable by the previous model, is a new host-parasite in vitro system (Leishmania mexicana amazonensis-J774G8 mouse macrophage line) developed by us recently. Not only can amastigotes be grown in continuous culture but also separated from host cell components with high purity and recovery. Therefore, we propose to employ these leishmania and macrophage materials for the following studies: (1) Nutritional biochemistry of heme requirement by intracellular leishmanias; (2) Characterization of parasite stage-specific protein molecules and their biosynthesis during this transformation; (3) Identification of parasite stage-specific surface antigens/glycoproteins in this transformation; and (4) Possible functions of these molecules in leishmania-macrophage binding, heme uptake or transport and resistance to macrophage lysosomal enzymes. Previously employed methodology will be followed for most of the proposed studies, i.e. (1) Fluorimetry of porphyrins and radioactive precursor incorporation experiments for heme and its biosynthesis; (2) S35methionine labeling-SDS PAGE-autoradiography for proteins and their biosynthesis; (3) Antibody-protein A sepharose/lectine agarose affinity binding of S35methionine labeled parasites followed by SDS PAGE-fluorography for their surface antigens/glycoproteins; and (4) Biological and radioactive assays already developed for studying the possible functions of these surface antigens or glycoproteins. The results of these studies will not only further our understanding of the basic mechanism of intracellular parasitism but also provide leads for developing effective chemo-or immuno-prophylaxis and therapy for leishmaniasis.